Characterization of Type VI secretion system in Edwardsiella ictaluri

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.

Edwardsiella ictaluri employs various defense mechanisms to adapt to intracellular stress.Among these mechanisms, the Type III Secretion System (T3SS) has been identified as crucial for replication within catfish macrophages [11].The secretion of T3SS effector proteins plays a vital role in facilitating bacterial replication within the macrophage environment [12,13].Notably, mutations in T3SS effector genes have been found to impair the intracellular replication of E. ictaluri [14].In addition to the T3SS, several genes have been identified as potentially significant for the survival of E. ictaluri inside neutrophils.These genes include enzymes involved in the tricarboxylic acid cycle (TCA), glycine cleavage system, sigmaE (σ E ) regulator, the SoxS oxidative response system, and a plasmid-encoded T3SS effector [15].
Initially, several T6SS proteins, including Eip19 (evpE), Eip18 (evpC), Eip55 (evpB), and Eip20 (evpA) were identified in E. ictaluri during catfish infection [22].The transcriptional regulation of the T6SS operon is governed by the Ara-C type regulatory protein EsrC, which is part of the EsrA-EsrB two-component system (TCs).EsrC acts as a sensor for environmental changes, such as variations in pH and inorganic phosphate (Pi) concentrations, thereby controlling the expression of the T6SS [11].Additionally, the transcription of evpP, a component of the T6SS, is regulated by the Ferric uptake regulator (Fur) protein, which binds to the Fur box in the evpP promoter [23].The T6SS in E. ictaluri serves a dual role, facilitating both survival within host cells and competition against inter-bacterial and intra-bacterial species.This versatility is achieved by delivering effector proteins into eukaryotic or prokaryotic cells [24].
The critical role of the T6SS in the Edwardsiella genus was initially demonstrated in the fish pathogen Edwardsiella piscicida [18,25].Secretion of EvpC, EvpI, and EvpP proteins has been observed, and mutations in T6SS genes, except evpD, resulted in attenuated virulence in the host [18].Evidence suggests a potential interaction between the secreted protein evpC and a disordered region of evpP, indicating that evpP may serve as a secreted effector protein primarily targeting inflammasome activation in macrophages [26,27].In E. ictaluri, evpP has been shown to induce increased necrosis in anterior kidney macrophages [28].The activation and repression of T6SS in E. piscicida are influenced by various environmental factors, including temperature, pH, Mg 2+ , P i , and iron availability [29,30].Our previous study revealed that hcp1 (evpC) and hcp2 are involved in virulence, adhesion to epithelial cells, and replication within catfish peritoneal macrophages, further emphasizing the importance of T6SS in the pathogenicity of E. ictaluri [31].
This study aimed to investigate the multifaceted role of the T6SS in E. ictaluri.Specifically, our objectives encompassed understanding the impact of T6SS on various important aspects, including survival and replication within macrophages, adhesion and invasion to catfish epithelial cells, adaptation and resilience to oxidative stress, as well as its contribution to virulence in catfish fingerlings and fry.By investigating the role of T6SS in the survival and replication of E. ictaluri within macrophages aids in unraveling the strategies employed by the bacterium to evade the host immune response and establish a persistent infection.This aspect highlights the interplay between the T6SS and the host's immune defense mechanisms.In addition, examining the involvement of T6SS in adhesion and invasion to catfish epithelial cells, we sought to elucidate the mechanisms through which E. ictaluri interacts with and establishes infection in host tissues.Understanding this aspect of the T6SS function provides valuable insights into the initial stages of pathogenesis.Furthermore, we aimed to explore the contribution of T6SS to the adaptation and survival of E. ictaluri under oxidative stress conditions.Oxidative stress is a significant challenge faced by bacteria during infection, and understanding the role of T6SS in oxidative stress resistance provides insights into the survival strategies of E. ictaluri within the host environment.Finally, assessing the impact of T6SS on the virulence of E. ictaluri in catfish fingerlings and fry enables us to establish the link between T6SS functionality and disease severity.By unraveling the specific contributions of T6SS to the pathogenicity of E. ictaluri, we can identify potential targets for therapeutic interventions and develop strategies to mitigate the impact of disease in aquaculture.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The protocol was approved by the Institutional Animal Care and Use Committee of the Mississippi State University (Protocol Number: 15-043).Catfish peritoneal macrophages were collected under tricaine methanesulfonate anesthesia (100 mg/ml), and all catfish used in the study were euthanized using a high dose of tricaine methanesulfonate (400 mg/ml).All efforts were made to minimize suffering.

Bacteria, plasmids, and growth conditions
Bacterial strains and the plasmid used in this work are listed in Table 1.The E. ictaluri strain 93-146 (EiWT) was cultivated at 30˚C either in brain-heart infusion (BHI) broth for 16 h or on BHI agar plates for 2 days.Escherichia coli CC118λpir and BW19851 (ΔuidA3::pir) strains were cultured in Lysogeny broth (LB) or on Lysogeny agar (LA) at 37 o C for 16 h.Antibiotics were incorporated into the culture medium, with final concentrations as follows: ampicillin (100 μg/ml), colistin (12.5 μg/ml), and gentamicin (10 μg/ml in macrophage culture or 100 μg/ ml to kill non-phagocyted E. ictaluri).Ampicillin and colistin were employed during the conjugation process.Ampicillin was used to select for E. ictaluri with the suicide plasmid, while colistin eliminated donor E. coli cells.Gentamicin was utilized in the bacterial killing assay to eliminate non-phagocytosed E. ictaluri selectively.

In-frame deletion of T6SS genes
The nucleotide sequences of the T6SS genes in E. ictaluri were acquired from the genomic data of E. ictaluri strain 93-146 [32].To generate T6SS mutants in E. ictaluri, splicing by overlap extension PCR was employed.In brief, external and internal primer pairs were designed to amplify the upstream and downstream regions of each target gene (Table 2).The resulting two fragments were merged through the process of splicing by overlap extension (SOEing) [33].The overlap extension PCR product was transferred to E. ictaluri by using plasmid pMEG375.Both the mutated insert (produced through overlap PCR) and pMEG375 underwent digestion with the same restriction enzymes, and the insert was subsequently ligated into pMEG375.Following electroporation and the selection of the correct plasmid in E. coli CC118, this plasmid was transferred to E. coli λpir strains via electroporation.These E. coli λpir strains were then utilized to transfer the plasmid into the E. ictaluri strain 93-146 through conjugation.A twostep selection process was implemented to obtain in-frame deletion mutants.In the first step, the conjugation mixture was inoculated in BHI broth containing ampicillin (selects for E. ictaluri with suicide plasmid) and colistin (eliminates donor E. coli).In the second step, positive colonies were streaked on BHI agar supplemented with colistin only.These colonies were restreaked on the BHI agar containing 5% sucrose, 0.35% D-mannitol, and colistin.The colonies that exhibited sensitivity to ampicillin and featured the mutant band were identified as inframe deletion colonies.The deletion of each gene was confirmed through PCR and sequencing.

Construction of bioluminescent strains
The creation of bioluminescent T6SS mutants followed a procedure previously documented [34].Briefly, E. coli SM10λpir harboring pAKgfplux1 and the mutant strains were cultured overnight and combined at a 1:2 ratio (donor: recipient).The resulting pellet was applied to a 0.45 μM filter paper on BHI agar and incubated at 30˚C for 24 h.The filter paper, containing a mixture of bacteria, was rinsed with BHI broth containing ampicillin and colistin, and serial dilutions were spread onto selective BHI agar containing ampicillin and colistin.Following incubation at 30˚C for 24-48 h, ampicillin-resistant mutant colonies carrying pAKgfplux1 emerged on the selective plates.

Bacterial killing assay
The bacterial killing assay was conducted following established procedures as outlined in previous references [31,[35][36][37].In brief, 1 ml squalene (Sigma-Aldrich) was administered to a year-old sedated (100 mg/ml tricaine methanesulfonate) channel catfish (ABW = 250-300 g) to activate catfish peritoneal macrophages.After 4 days of injection, sterile and cold phosphate-buffered saline (PBS) was injected into the intraperitoneal area, and the cell suspension was collected into tubes on ice.Additional PBS was injected into the peritoneal cavity until the fluid became clear, and subsequently, the catfish were humanely euthanized (400 mg/ml tricaine methanesulfonate).Peritoneal macrophages (5 x 10 5 cells) were harvested and mixed with bioluminescent E. ictaluri strains at a 1:1 ratio in a 96-well plate (Evergreen Scientific).
Each well contained a final volume of 200 μl for the cell-bacteria mixture, and the plate included four replicate wells for each treatment and a negative control (containing only cells).
The plate was centrifugated at 1500 rpm for 5 minutes at 24˚C to compact the cells and bacteria at the bottom.Subsequently, the plate was incubated for 1 h at 30˚C to facilitate the invasion of catfish peritoneal macrophages by bioluminescent mutants and EiWT.After this initial incubation, the cell-bacteria mixture underwent centrifugation at 2000 rpm for 7 min, and the culture medium was aspirated.Fresh medium containing 100 μg/ml gentamicin was added, and the cells were further incubated for 1 additional h at 30˚C to eliminate non-phagocyted E. ictaluri.Following this, each well was subjected to triple washes with PBS, and the peritoneal macrophages were resuspended in Channel Catfish Macrophage Medium supplemented with 10 μg/ml gentamicin.Finally, cells were transferred to black 96-well plates (Fisher Scientific), and the plate was placed in Cytation 5 instrument (BioTek), where the cells were incubated for 48 h under 5% CO 2 at 30 o C. Bioluminescence data were recorded at hourly intervals, and subsequent analysis was performed to determine the number of surviving bioluminescent bacteria within catfish peritoneal macrophages.

Attachment and invasion assays
Attachment and invasion assays were conducted using previously established procedures with some adjustments [38].Briefly, channel catfish ovary (CCO) cells were suspended in Dulbecco's Modified Eagle Medium (DMEM) (Sigma), supplemented with 10% fetal bovine serum and 4 mM L-glutamine, resulting in a final concentration of 1 x 10 7 cells per ml.Bioluminescent mutants and EiWT were combined with CCO cells at a 1:1 ratio and seeded in a 24-well plate.Each well held a final volume of 1 ml cell-bacteria mixture, and the plate included four replicate wells for each treatment, alongside a negative control with no bacteria.The plate was incubated at 28˚C for 1 h to facilitate the attachment of mutants and EiWT to CCO cells.Subsequently, the cell suspensions were exposed to DMEM containing 100 μg/ml gentamicin for 1 h to eliminate the externally located bacteria.The plate underwent three washes with PBS, and the bioluminescent emissions originating from the E. ictaluri strains were captured using the IVIS Lumina XRMS in Vivo Imaging System Series III and quantified utilizing Living Image Software version 4.7.4 (PerkinElmer).

Stress assays
The resilience of the mutants to oxidative stress induced by hydrogen peroxide (H2O2) (Sigma) and nitrosative stress caused by sodium nitroprusside (SNP) (Sigma) was evaluated in both a rich medium (BHI) and a low phosphate minimal medium at pH 5.5 (MM19-P) [39].Bacterial cultures were cultivated overnight, and OD 600 was standardized to 0.5 for each culture.Subsequently, five μl of bacteria from each strain were introduced into 195 μl of BHI and MM19-P broth, each containing either 0.75 mM H 2 O 2 (prepared from 30% stock solution) or 5 mM SNP.A 96-well black plate was employed for each stress condition, with three replicates allocated for each mutant, along with EiWT as a positive control.After 4, 8, 12, and 24 h of incubation at 30˚C, the average photon counts were determined using the IVIS Lumina XRMS in Vivo Imaging System Series III (PerkinElmer).

Virulence and efficacy of T6SS mutants in catfish fingerlings and fry
Vaccination and efficacy assessments were conducted following previously established procedures [20].In the vaccination stage, catfish are infected with mutants to determine their attenuation levels and vaccinate them.In the challenge stage, vaccinated catfish (catfish that survived after mutant infection) are infected with E. ictaluri wild-type (EiWT) 21 days postvaccination to assess efficacy.Briefly, specific-pathogen-free (SPF) channel catfish fingerlings and fry were obtained from the MSU-CVM Hatchery.Twenty-five catfish fingerlings (10.46 ± 0.86 cm, ABW = 14.03 ± 3.57 g) were stocked into each tank and acclimated at 26-28˚C for one week.During acclimation and experiments, 12 h on and 12 h off light cycle was applied, catfish were fed twice a day, and chlorine, dissolved oxygen, and temperature were monitored daily.Tanks were randomly assigned to 13 T6SS mutants (vaccination), EiWT (positive control), and BHI (sham) groups.Each treatment had three replicates.Immersion vaccination was applied by lowering the water level in each tank to 10-L and adding 100 ml of bacterial culture (final dose of 2.4 x 10 7 CFU/ml water).After 1 h, water flow (1 liter/min) was restored to each tank.Infected fish were monitored twice a day, and dead fish were collected.In addition, fish exhibiting loss of equilibrium accompanied by disease-specific clinical signs, such as petechial hemorrhages, exophthalmia, swollen abdomen, and skin lesions, were removed immediately and euthanized (400 mg/ml tricaine methanesulfonate) to minimize suffering.Mortalities were recorded daily for 21 days, and the percent mortalities were calculated for each group.To assess the protective capabilities of mutants, all fish that survived the vaccination were challenged with EiWT (2.8 x 10 7 CFU/ml) 21 days post-vaccination, as described above.The experiment was terminated by euthanizing all fish using 400 mg/ml tricaine methanesulfonate when no fish mortalities were observed for three consecutive days.Virulence and efficacy of mutants exhibiting good attenuation and protection in fingerlings were tested in 14-day-old catfish fry (0.14 cm, ABW = 0.03 g) as described above.

Statistical analysis
The significance of variations in means among treatment groups was determined using oneway ANOVA and two-way ANOVA procedures, followed by Tukey's test for post hoc analysis.These analyses were conducted within the SAS for Windows 9.4 software (SAS Institute, Inc., Cary, NC).A significance threshold of p < 0.05 was adopted for all tests.

Components of E. ictaluri T6SS
E. ictaluri harbors a compact T6SS operon spanning 20,784 nucleotides, encompassing the evpP to evpO genes (Fig 1).Notably, in addition to this operon, an hcp2 gene, which shares homology with evpP, was identified outside of the core T6SS operon.

Phagocytic uptake and survival of the mutants in catfish peritoneal macrophages
Uptake and intracellular survival of the T6SS mutants and WT within catfish peritoneal macrophages were summarized in Fig 2 .In phagocytic uptake, EiΔevpF, EiΔevpG, EiΔevpI, EiΔevpJ, and EiΔevpL showed significantly higher internalization rates than other T6SS mutants and EiWT at 0 h.At 6 hours, the numbers of intracellular EiΔevpE, EiΔevpF, EiΔevpG, EiΔevpI, EiΔevpJ, EiΔevpK, and EiΔevpL were significantly higher than EiWT.EiΔevpF and EiΔevpG replicated in significantly higher rates than other mutants, and interestingly, EiΔevpO remained at a lower replication rate than EiWT.EiΔevpE, EiΔevpF, EiΔevpG, EiΔevpJ, and EiΔevpK survived inside the catfish peritoneal macrophages up to 12 h.EiΔevpL had a higher phagocytic uptake rate at 0 h, but the number of intracellular EiΔevpL decreased after 6 h.Although EiΔevpE and EiΔevpK had similar phagocytic uptake as EiWT, the number of intracellular EiΔevpE and EiΔevpK increased up to 12 h.At 24 h, the number of intracellular T6SS mutants and EiWT were significantly decreased, and there were no significant differences among T6SS mutants and EiWT.Together, these data indicated that EiWT could replicate in catfish peritoneal macrophages for a limited time (6 h), and after this, macrophages kill EiWT gradually.Deleting T6SS genes caused an increased uptake of some mutants by macrophages at 6 h, but the killing of mutants progressed at a pattern similar to EiWT.

Attachment and invasion of the mutants in CCO cells
Attachment and invasion of T6SS mutants and EiWT were assessed in CCO cells by bioluminescent imaging (Fig 3A

Survival and stress resistance of the mutants
Innate immune cells can respond to pathogenic bacteria invasion by activating nitrosative and oxidative stress mechanisms.SNP and H 2 O 2 stresses were applied to imitate the phagosomal stress conditions.The minimal medium (MM19-P) mimicked the nutrient-poor phagosomal conditions, in which T6SS mutants showed lower growth than BHI (Figs 4A and 5A).At 0-12 h, some T6SS mutants showed variable resistance to SNP in BHI, but growth of EiWT was higher than all mutants at 24 h (Fig 4B -4F).Similarly, T6SS mutants showed variable resistance to SNP in MM19-P at 0-12 h, but growth of EiΔevpI and EiΔevpK was higher than all mutants and EiWT (Fig 5B -5F).The treatment of T6SS mutants with H 2 O 2 caused reduced bacterial growth in both BHI and MM19-P (Figs 6A and 7A).Growth of EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO was significantly affected by H 2 O 2 stress in BHI, and at 24 h, growth of all mutants except EiΔevpD was lower than EiWT (Fig 6B -6F).In MM19-P, H 2 O 2 stress caused significant growth loss for EiΔevpA, EiΔevpH, EiΔevpI, EiΔevpK, EiΔevpM, EiΔevpN, and EiΔevpO by 24 h (Fig 7B -7F).These results show that T6SS mutants are more sensitive to SNP and H 2 O 2 in MM19 due to the low pH (5.5) in MM19-P.The resistance of T6SS mutants to SNP is more than H 2 O 2 .Hydrogen peroxide can restrict the bacterial growth of T6SS mutants in both MM19-P and BHI.T6SS mutants are sensitive to stress factors that imitate phagosomal killing conditions inside catfish macrophages.

Discussion
This research involved the development of T6SS mutants, and their performance in various aspects was thoroughly examined.These aspects encompassed their ability to survive and replicate within catfish peritoneal macrophages, their attachment and invasion capabilities in catfish epithelial cells, their resilience against stressors, as well as their virulence and effectiveness in catfish fingerlings and fry.Please refer to Table 3 for a summary of the findings.
The T6SS in E. ictaluri is composed of an operon containing the genes evpP to evpO and an additional hcp2 gene, a homolog to evpP, outside the operon (Fig 1).The entire T6SS operon in E. ictaluri spans a total of 20,784 nucleotides.Although multiple copies of the T6SS is not observed in E. ictaluri, presence of functionally non-redundant multiple copies of the T6SS in bacterial genomes and existance of plasmid encoded T6SS have been reported [40].
Survival within-host immune cells is crucial for E. ictaluri as a facultative intracellular pathogen.Studies on other bacteria, such as Bordetella bronchiseptica and Salmonella enterica, have demonstrated that deleting T6SS genes, such as tssH (clpV) and tssM, enhances intracellular replication [41,42].However, in the case of E. ictaluri, it has been observed that mutations in tssE, tssM, and tssH lead to a decrease in intracellular proliferation inside macrophages [43][44][45][46].The uptake of EiΔevpF (tssF), EiΔevpG (tssG), EiΔevpI (tssI), EiΔevpJ (tssJ), and EiΔevpL (tssL) inside peritoneal macrophages was higher than the other T6SS mutants in E. ictaluri (Fig 2).Among these mutants, EiΔevpF, EiΔevpG, and EiΔevpJ increased uptake in peritoneal macrophages.On the other hand, all T6SS mutants and EiWT could not replicate in peritoneal macrophages.This could potentially be attributed to the activated state of catfish peritoneal macrophages, which may limit the intracellular replication of E. ictaluri.It is worth noting that while deletion of tssM and tssH resulted in different effects on the fitness and survival of several  underscoring the critical role of the T6SS as an essential secretion system for E. ictaluri's adaptation to catfish peritoneal macrophages.
Studies have provided compelling evidence supporting the role of the T6SS in host cell adherence and invasion.Disruption of tssM reduced T6SS-mediated adhesion and invasion in various bacteria, including E. coli, Campylobacter jejuni, and Vibrio parahaemolyticus [44,47,48].Interestingly, mutation of tssM in Helicobacter hepaticus resulted in increased adhesion [49].EiΔevpO homolog of tssM has significantly reduced ability in attachment and invasion for CCO cell lines (Fig 3).Similarly, EiΔevpN (tssL) showed significantly less attachment and invasion of the host epithelial cells.Loss of vgrG can also affect the attachment and invasion of bacteria for host epithelial cells [50].EiΔevpI, a homolog of vgrG, had no significant difference with EiWT regarding attachment and invasion for CCO.Remarkably, EiΔevpF (tssF), EiΔevpG (tssG), and EiΔevpL (tssJ) were significantly more adhesive and invasive than EiWT.Although EiΔevpK (tssA) had more attachment ability, its invasion rate was limited.These findings highlight the diverse effects of T6SS mutations on the attachment and invasion abilities of E. ictaluri in host epithelial cells and underscore the complex interplay between the T6SS and hostpathogen interactions in E. ictaluri.
The role of the T6SS in stressful conditions highlights its potential importance in the acquisition of crucial metals necessary for bacterial survival within the host [51].These metals, such as manganese, play a vital role in countering oxidative stress conditions observed in Burkholderia thailandensis [52].Once internalized by host macrophages, bacteria face various stress and killing mechanisms activated by the host to restrict intracellular replication [53].However, E. ictaluri has evolved mechanisms to resist these stress and killing mechanisms by upregulating the expression of specific genes [54].In our study, most T6SS mutants subjected to treatment with SNP and H 2 O 2 in BHI and MM19-P media (Figs 4-7) showed limited growth under H 2 O 2 -induced stress conditions in both BHI and MM19-P (Figs 6 and 7).Notably, the growth of EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO mutants was defective in both BHI and MM19-P media.This outcome strongly suggests that T6SS plays a critical role in enabling the survival of E. ictaluri under stressful conditions.Deletion of specific T6SS mutants may significantly impact the ability of E. ictaluri to withstand stress conditions that simulate the phagosomal environment of macrophages.These findings underscore the importance of the T6SS in conferring stress resistance and enhancing the survivability of E. ictaluri in challenging host environments.The ability to counteract oxidative stress and other stressors encountered within the host is crucial for the pathogen's persistence and successful establishment of infection.
The loss of a functional T6SS can result in a significant reduction in the virulence of pathogenic bacteria.Deletion of tssM in Aeromonas hydrophila, C. jejuni, and Acinetobacter baumannii has been shown to cause attenuation in mouse models, indicating the crucial role of T6SS in their pathogenicity [47,55,56].Similarly, in E. piscicida evpH, evpI, and evpC were found to be essential for establishing an infection in blue gourami [18].In our study, we investigated the virulence of T6SS mutants in catfish fingerlings and fry.Our data revealed a clear trend: mutants that exhibited complete attenuation in fingerling fish also displayed high levels of attenuation in catfish fry, while mutants with less attenuation in fingerling fish were found to be highly virulent in catfish fry (Fig 8).This consistent observation suggests the reliability and reproducibility of our experiments.Additionally, it highlights the fact that catfish fry possess a less developed immune system compared to fingerling fish [57], rendering them more susceptible to bacterial infections.These findings further support the critical role of the T6SS in the virulence of E. ictaluri.Deletion of T6SS genes in our study resulted in a significant reduction in virulence in both catfish fingerlings and fry, emphasizing the importance of this secretion system in the pathogenesis of E. ictaluri.

Conclusions
In conclusion, this study has elucidated the multifaceted role of T6SS in E. ictaluri.Firstly, it was revealed that T6SS mutants exhibited varying behaviors in terms of survival and replication within host immune cells, with mutations in tssE, tssM, and tssH leading to a decrease in intracellular proliferation, underscoring the critical role of the T6SS in adaptation to catfish peritoneal macrophages.Secondly, the study demonstrated the intricate relationship between the T6SS and host cell adherence and invasion, with different mutants displaying contrasting abilities in attachment and invasion of host epithelial cells, indicating the complex interplay in host-pathogen interactions.Thirdly, the T6SS was shown to be pivotal in enhancing bacterial survival under stressful conditions, particularly oxidative stress, highlighting its importance in E. ictaluri's ability to withstand host defenses.Lastly, the study unequivocally established the indispensable role of the T6SS in the virulence of E. ictaluri, with mutants displaying reduced virulence in catfish fingerlings and fry, shedding light on its critical contribution to pathogenesis.These collective findings underscore the significance of the T6SS as an essential secretion system for E. ictaluri's adaptation, survival, and pathogenicity within the catfish host.The implications of these results extend beyond understanding the basic biology of E. ictaluri to potentially providing insights for developing effective strategies to control and prevent infections in catfish populations, thus contributing to the broader field of aquaculture and fish health.The diversity of effects observed in this study underscores the complexity of T6SS and its crucial, multifaceted role in the intricate host-pathogen interactions of this bacterium.

Fig 4 .Fig 5 .
Fig 4. Stress assays of T6SS mutants and EiWT exposed to SNP in BHI.The mean photon emission was obtained from each mutant and positive (EiWT) and negative (no bacteria) controls.The data represent the mean ±SD from one experiment.The experiment included three replicate wells for each mutant and control.Negative control was used to subtract background noise bioluminescence from each sample.The lowercase letters show significant differences between treatments (p < 0.05).(A) Bioluminescent image of T6SS mutants and EiWT exposed to SNP in BHI for 24 h.(B -F) The bar graphs indicated the relative luminescence unit (RLU) obtained from the T6SS mutants and EiWT treated with SNP in BHI for 0, 4, 8, 12, and 24 h.https://doi.org/10.1371/journal.pone.0296132.g004

Fig 6 .
Fig 6.Stress assays of T6SS mutants and EiWT exposed to H 2 O 2 in BHI.The mean photon emission was obtained from each mutant and positive (EiWT) and negative (no bacteria) controls.The data represent the mean ±SD from one experiment.The experiment included three replicate wells for each mutant and control.Negative control was used to subtract background noise bioluminescence from each sample.The lowercase letters show significant differences between treatments (p < 0.05).(A) Bioluminescent image of T6SS mutants and EiWT exposed to H 2 O 2 in BHI for 24 h.(B -F) The bar graphs indicated the relative luminescence unit (RLU) obtained from the T6SS mutants and EiWT treated with H 2 O 2 in BHI for 0, 4, 8, 12, and 24 h.https://doi.org/10.1371/journal.pone.0296132.g006

Fig 7 .
Fig 7. Stress assays of T6SS mutants and EiWT exposed to H 2 O 2 in MM19-P.The mean photon emission was obtained from each mutant and positive (EiWT) and negative (no bacteria) controls.The data represent the mean ±SD from one experiment.The experiment included three replicate wells for each mutant and control.Negative control was used to subtract background noise bioluminescence from each sample.The lowercase letters show significant differences between treatments (p < 0.05).(A) Bioluminescent image of T6SS mutants and EiWT exposed to H 2 O 2 in MM19-P for 24 h.(B -F) The bar graphs indicated the relative luminescence unit (RLU) obtained from the T6SS mutants and EiWT treated with SNP in MM19-P for 0, 4, 8, 12, and 24 h.https://doi.org/10.1371/journal.pone.0296132.g007

Fig 8 .
Fig 8. Attenuation, vaccination, and efficacy testing of T6SS mutants.In the vaccination stage, fish are infected with mutants to determine their attenuation levels and, at the same time, vaccinate fish.In the challenge stage, vaccinated fish (fish survived after mutant infection) are infected with EiWT 21 days post-vaccination to assess efficacy.(A) virulence and (B) efficacy of T6SS mutants and EiWT in catfish fingerlings.(C) virulence and (D) efficacy of T6SS mutants and EiWT in catfish fry.The data represent the mean percent mortalities ±SD from one experiment.The experiment included three replicate fish tanks, each containing 25 fish.The letters above bars show statistical significance between treatments (p < .05).https://doi.org/10.1371/journal.pone.0296132.g008

Table 3 . Mutants of T6SS genes in E. ictaluri.
Summary of results: This table provides an overview of the outcomes in various aspects, including survival and replication within catfish peritoneal macrophages, attachment and invasion capabilities in catfish epithelial cells, resistance to stress factors, and virulence and efficacy in catfish fingerlings and fry.Definitions of abbreviations or acronyms used: BKA: Bacterial killing assay, involving peritoneal macrophages, uptake at 0 h and survival at 6 h CCO: Channel catfish ovary cells, representative of epithelial cells, Stress Assays: Cells subjected to stress factors at 8 hours, SNP: Sodium nitroprusside, inducing nitrosative stress, H 2 O 2 : Hydrogen peroxide, causing oxidative stress, BHI: Brain-heart infusion broth, a nutrient-rich medium, MM19-P: Low-phosphate minimal medium broth at pH 5.5, a minimal medium, Virulence: Evaluation through catfish immersion challenge with mutants and EiWT after 21 days, Efficacy: Assessment via immersion re-challenge of vaccinated catfish with EiWT after 21 days, Fingerling: Refers to six-month-old catfish, Fry: Pertains to two-week-old catfish.(+) indicates a significant difference compared to EiWT (p < 0.05) and (-) denotes no significant difference compared to EiWT (p < 0.05).N/A: Abbreviation for "Not Applicable".https://doi.org/10.1371/journal.pone.0296132.t003